The past several decades have seen tremendous advances in our understanding of biological processes, and the rational design of pharmaceutical drugs based upon knowledge of those processes. Much effort has been devoted to identifying natural and synthetic ligands capable of interacting with proteins involved in biological processes. The recognition domains of proteins control biological processes in many ways, including: serving as loci of catalytic activity; allostearically regulating enzymatic activity; mediating signal transduction; effecting transport of cellular components; activating factors involved in transcribing or translating nucleic acids; and many others.
Drug discovery today typically follows the following sequence: (1) an active molecule is identified and compared to other molecules that have or do not have relevant biological activity; (2) based upon structure/activity relationships between the molecules, a chemist designs and prepares a library of potentially active molecules, often using combinatorial techniques; (3) the molecular biologist screens the library of molecules for relevant biological activity; (4) based upon the results of this screen, the chemist might prepare another library of compounds for testing. This process is repeated until several lead compounds are identified for more detailed investigation.
The foregoing method has led to the discovery of many important drugs. However, even high volume combinatorial chemistry and biological screening methods have proven no match for the complexity of protein structures and protein/ligand interactions involved in various biological mechanisms. A molecule might exhibit a relevant biological property in vitro, which scientists would attribute to ligand binding to a relevant protein. However, if the synthetic ligand does not sufficiently mimic the natural ligand, it can do more harm than good by affecting biological processes that are unrelated to the disease state being treated. In addition, if the synthetic ligand does not bind to the protein with sufficient affinity, it will not regulate the protein's activity sufficiently to control the targeted biological process.
The complexity of proteins greatly inhibits our ability to design custom synthetic ligands. Protein recognition domains often consist of multiple binding sites spread over different regions of the protein. Moreover, because the binding sites on proteins have specific three dimensional conformations, binding components of a ligand must be properly oriented to match the protein binding sites to have the appropriate effect. Variations in noncovalent interactions between ligands and proteins, such as the balance between Van der Waals and electrostatic interactions, further complicate the ability to design a synthetic ligand for a protein.
Efforts have been made recently to use NMR to improve the binding affinity of known ligands, by screening for other ligands that bind a protein near the protein binding site for the known ligand. Once a suitable second ligand is identified, the ligands are covalently linked to construct a hybrid ligand having greater binding affinity for the protein than either of the constituent ligands. A series of patents to Fesik et al., U.S. Pat. Nos. 5,698,401; 5,804,391; 5,891,643; 5,989,827; and 6,043,024 (“the Fesik patents”) disclose such efforts, through a technique known as “SAR by NMR.”
SAR by NMR uses a very sensitive two dimensional NMR experiment, a heteronuclear single quantum coherence (HSQC) experiment, to screen compound libraries for components that bind to protein targets, and uses a mapping of perturbed peaks to points in a three dimensional protein structure from the HSQC experiment to locate sites of binding on a protein surface. The experiment relies on uniform 15N enrichment of the protein target and collection of peaks that correlate the 1H and 15N chemical shifts of directly bonded 15N—1H pairs that occur primarily in backbone amide bonds of the protein, one pair per residue. Effects on the chemical shifts of peaks coming from amide pairs on binding of drug components is largely restricted to proximate residues, and thus provides qualitative information on the location of the binding site for any one component. If the peaks can be assigned to specific amino acids and if the protein structure is known, the binding site can be spatially localized. When more than one interacting component can be localized, components binding to proximal sites can be assembled synthetically to achieve binding affinities that approximate the product of the individual component affinities. Thus, compounds that individually fail as drug leads because of low binding affinities can be combined to produce viable leads.
The SAR approach, while successful, is limited. The procedure does require assignment of peaks to the amino acid sequence of the protein, and it does require knowledge of the three dimensional structure of the protein. It is also often the case that additional experiments involving nuclear Overhauser effects (NOEs) between protons on a binding component and protons on the protein are needed to restrict possible orientations of each binding component relative to the protein surface and better define the relative geometries of components to be linked synthetically. Thus, even though the basic HSQC screen experiment is highly efficient, the additional experiments needed for assignment and structure determination are very time consuming. They also begin to fail when proteins become large. Work to date has been restricted to proteins that are less than 40 kDa in molecular weight and soluble to levels approaching 0.5 mM.
SAR by NMR is further limited by the intrinsic limitations of nuclear Overhauser effects. For example, to observe NOEs between a ligand and a protein in a complex, the NMR observable protons on the ligand must be sufficiently close to NMR observable protons on the protein surface for NOEs to be measured (typically within 5 Å). NOEs also rarely are used to characterize some types of complexes, for example oligosaccharide-protein complexes. In the latter case it is difficult to characterize interactions because the hydrogen-bonding networks involving hydroxyl proteins on the sugars are often part of the interface between protein and oligosaccharide. The hydroxyl protons are then the bulk of ones within 5 Å of protein protons, but they exchange rapidly with protons in bulk water making their NMR resonances hard to observe.
Therefore, it is an object of this invention to provide a method of identifying two or more ligands that bind to a protein recognition domain without assigning NMR peaks to the amino acid sequence of the protein, or characterizing the 3-D structure of the recognition domain.
It is another object of the invention to estimate the distance between ligand binding sites of the identified ligands on the surface of the protein.
It is another object of the invention to identify two or more ligands that bind to a biological target, and to covalently link the ligands at a bond length that approximates the distance of separation between the ligands when bound to a biological target, to thereby produce hybrid ligands having improved binding affinity for the biological target.
It is still another object of the invention to ascertain three dimensional orientations of ligands when bound to a biological target, and to combine the orientational information with distance information to produce hybrid ligands having even greater binding affinity for a biological target.
It is a further object of this invention to provide methods for characterizing ligand/protein interactions not susceptible to characterization by NOEs and other traditional NMR methods.